Stable and bioavailable compositions of isomers of lycopene for skin and hair

ABSTRACT

The present invention relates to a method of manufacturing a stable composition enriched in cis-lycopene (z-isomers) by prolonged heating in solvents of tomatoes, parts of tomatoes, derivative thereof or tomato extracts in solvents.

The present invention relates to a primary composition that includes atleast one lycopene-containing material enriched in Z-isomers of thelycopene compound having an increased stability and bioavailability, andprocess of forming the same. It also relates to an oral composition thatcontains the primary composition in a foodstuff, in a food supplement,in a cosmetic preparation or in a pharmaceutical preparation.

TECHNOLOGICAL BACKGROUND

Absorption of carotenoids is a complex process involving release fromthe food microstructure matrix, dissolution into mixed micelles,intestinal uptake, incorporation into chylomicrons, distribution to thetissues, uptake by liver and re-secretion into VLDL, which areprogressively transformed into LDL.

Lycopene absorption from food sources is widely documented. Lycopenebioavailability is quite low from foods such as tomatoes and tomatojuice. Up to now, tomato paste is the best known food source forbioavailable lycopene. Tomato contains about >90% of lycopene in its allE configuration.

Tomato extracts containing a high amount of lycopene are commerciallyavailable in the form of oleoresin but the bioavailability of thelycopene in humans is rather limited from these sources. In concentratedtomato extracts, lycopene is mainly present in crystalline form, whichhas been suggested to be one of the primary factors that reduces itsbioavailability.

To date, most commercially available lycopene sources display anisomeric profile quite similar to the starting tomatoes or show only aslight increase in Z-isomers, whether they are derivatives (such assauces) or extracts. A number of treatments, as for instance thermalprocessing, are known to promote isomerization. Shi et al., Journal ofFood Process Engineering 2003, 25, 485-498, showed that an increase in Zisomers could be obtained by heating tomato sauces. However certainlycopene isomers are not stable and prone to retro-isomerisation.According to the literature, 5-Z is the most stable among thepredominant lycopene isomers followed by the all-E, the 9-Z and the13-Z. Accordingly, the stability of isomerised lycopene based productsdepends on their lycopene isomer profile and thus can be modulated bytechnological processings affecting this profile.

Thermal isomerisation of lycopene is known to improve itsbioavailability from food matrices. However, the bioavailability ofindividual lycopene isomers has not been investigated yet. As forstability, it can be assumed that bioavailability of lycopene basedproducts is dependent on their lycopene isomer profile and thereby canbe modulated by technological means.

There are already patents that propose technological means andformulations for improved bioavailibility of lycopene. For example, WO2005/075575 provides a primary composition enriched in Z-isomers,effective to increase the bioavailability of lycopene.

EP 1 103 579 discloses a process for extracting lycopene by refluxingethanol: the process is carried out for short time (30′) so that nosignificant isomerization occurs. Actually, the natural all-Z lycopeneis desired as the final product.

WO 96/13178 discloses a process for the preparation of stable lycopeneconcentrates: the effect of isomerization on stability is not howeverappreciated and, on the contrary, the disclosed process tries to avoidisomerization by using temperatures lower than 50° C. for a few minutes,e.g. 10 minutes.

EP 1 201 762 refers to a lycopene-containing product wherein lycopene isin its natural all-trans form. No significant isomerisation takes placesince any heat treatment (dissolution and concentration) is carried outfor the shortest possible time (less than 1 hour).

KR 2005 006592 discloses the preparation of a lycopene-zinc complexhaving increased anti-oxidant activity. Hating is applied in the absenceof solvents to break the cell walls of the plant material. No prolongedheating and no isomerization occurs.

WO 03/079816 discloses a process for the preparation of tomato extractswith high content in lycopene wherein the extraction is carried out atroom temperature.

U.S. Pat. No. 5,837,311 and WO 97/48287 disclose a process for thepreparation of an oleoresin having high content of lycopene andsatisfactory stability. The latter feature is obtained by means ofphospholipids and glycerides present in the oleoresin itself. No mentionis made about the isomeric composition of lycopene and of its influenceon the stability. An hot extraction is carried out in order to maximisethe yield in lycopene but for times not longer than 1.2 hours. A heatpre-treatment is carried out on the tomatoes in the absence of anysolvent only in order to improve the pulp-serum separation.

WO 2005/075575 addresses the problem of increasing the cis-isomerscontent in tomato oleoresins: isomerization is obtained, inter alia, bythermal treatment for short period of time. Moreover, the obtainedproduct contains an high amount of the unstable 13-cis isomer.

EP 0 937 412 discloses a process for the preparation of finely dividedpulverous carotenoid preparations including an heating step in thepresence of solvents for very short times (5 seconds).

U.S. Pat. No. 5,858,700 concerns a process for the isolation andpurification of lycopene crystals characterised by the hydrolysis ofimpurities such as glycerides and phosphonates. The hydrolysis iscarried out at high temperature but for times shorter than 2 hours,optionally preceded by an extraction step with refluxing solvents, butalways for short time in order to prevent the lycopene degradation.

U.S. Pat. No. 6,235,315 discloses stable, pulverulent lycopeneformulations characterised in that lycopene has a certain degree ofcrystallinity and the lowest possible degree of isomerization.

WO 03/090554 discloses concentrated tomato derivatives having highcontent of lycopene and pre-determined viscosity and sugar content. Theprocess is physical and no solvent is used. Heating for 1-6 minutes IIis carried out on round tomatoes in order to make the pulp separationeasier.

Mayer-Miebach at al., “Thermal processing of carrots: lycopene stabilityand isomerisation with regard to antioxidant potential” Food ResearchInternational, Elsevier Applied Science, Barking, GB, vol. 38, no. 8-9,October 2005, pages 1103-1108, study the isomerisation of lycopene infreeze-dried carrots in function of temperature. The Authors concludethat only the all-trans isomer is stable to prolonged heating, resultingin a decrease of cis-isomers.

As a matter of fact, therefore, the prior art does not provideisomerisation processes of lycopene comprising a prolonged heating inorganic solvents allowing to obtain a product having highbioavailability and stable isomeric composition.

SUMMARY

It has been found that the stability of individual Z-lycopene isomersvaries from one isomer to another; in particular the 13-Z lycopene wasmuch less stable than either the 5-Z, or the 9-Z, or the all-E isomers.Consequently, a primary composition according to the present inventionmust have a level of 13-Z isomer as low as possible to exhibit optimalstability. It has also been shown that some Z isomers (such as 5-Z and9-Z, for example) of lycopene enhance the bioavailability of thecomposition containing lycopene. The primary composition must thereforecontain mainly the 5-Z isomer, or a combination of 9-Z and 5-Z isomersto provide an improved bioavailability and bioefficacy.

Accordingly, it is a first object of the present invention to provideprimary compositions with at least one lycopene-containing materialenriched in a specific mixture of Z isomers of lycopene, thelycopene-containing material containing by weight a greater percentageof an isomer selected from the group consisting of 5-Z, 9-Z andcombinations thereof than of 13-Z isomer.

In an embodiment, the present invention provides an oral compositionthat contains the primary composition in a foodstuff, in a foodsupplement, in a cosmetic preparation or in a pharmaceuticalpreparation.

In an embodiment, the present invention provides the primary compositionas an additive in a foodstuff for oral administration, such as in anutritional composition, a food supplement, a pet food product, acosmetic preparation or a pharmaceutical preparation.

In an embodiment, the present invention provides a method ofmanufacturing the primary compositions or food supplements, cosmeticpreparations or pharmaceutical preparations containing the same.

In another embodiment, the present invention provides the use of theprimary composition as described above, for the preparation of an oral,cosmetic or pharmaceutical composition intended for improving skinhealth, in particular for photoprotection of the skin or for protectingskin tissue against aging.

In an alternative embodiment, the present invention provides the use ofthe primary compositions for the preparation of an oral, cosmetic orpharmaceutical composition for preventing or treating cardiovasculardiseases or cancers.

An advantage of the present invention is to provide compositions of Zisomers of lycopene that exhibit a higher stability, bioavailability andbioefficacy.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description and the FIGURE.

BRIEF DESCRIPTION OF THE FIGURE

FIGURE. Area under the curve (AUC) of plasma lycopene/triglycerides ofTRL following the consumption of a standard meal containing 25 mg totallycopene from either tomato paste (all-E lycopene) or tomato oleoresinrich in 5-Z lycopene (5-Z oleoresin) or tomato oleoresin rich in 13-Zlycopene (13-Z oleoresin) or tomato oleoresin rich in 9 and 13-Zlycopene (9- & 13-Z oleoresin).

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to compositions that providehealth benefits. More specifically, the present invention relates tobeneficial nutritional compositions that can be used to improve skin andhair and methods regarding the same.

The present invention now makes available to the consumer an improvedcomposition obtained from natural products. The primary compositionprovides lycopene in a particularly highly bioavailable and/orbioeffective form.

In a preferred embodiment, the invention provides tomato extracts orderivatives thereof with an isomer ratio different from the naturallyoccurring one in products to date available. In particular, theinvention relates to extracts or derivatives with an E isomer contentnot higher than 60% on total lycopene content, preferably with an Eisomer content not higher than 40% on total lycopene content (by HPLC).

In an embodiment, the present invention provides a primary compositioncontaining a specific combination of Z isomers. Preferably, the ratio ofZ/E isomers in the primary compositions of the present invention shouldbe above 1.

Moreover, the combination is preferably rich in 5-Z and 9-Z and poor in13-Z. In a preferred embodiment, the amount of 5-Z and 9-Z is greaterthan 30% on total lycopene content, preferably greater than 40%, mostpreferably greater than 50%. Also, the amount of 13-Z is less than 10%on total lycopene, preferably less than 5%, most preferably less than 3%on total lycopene content. By increasing the specific 5-Z and 9-Zisomers and/or decreasing the 13-Z isomers, for example, a stable formof the primary composition that is more bioavailable and morebioeffective can be obtained. Moreover, the extracts or derivatives ofthe invention are stable under the usual storage conditions and do notundergo retro-isomerization. Under ordinary protective conditions(absence of light and oxygen), the lycopene total content and E isomercontent remains constant. The latter does not increase, even whenkeeping the extracts at room temperature.

Such a profile (i.e. low amount of 13-Z isomer of the lycopene) may befor example obtained by isomerizing lycopene using catalysis on a solidmatrix such as clays, or by prolonged heating.

In an embodiment, the lycopene-containing material can be, for example,in the form of an extract, a concentrate or an oleoresin. In the presentspecification, the term “oleoresin” should be understood to mean a lipidextract of a lycopene-containing material, which includes possibly othercarotenoids, triglycerides, phospholipids, tocopherols, tocotrienols,phytosterols and other less significant compounds. It has beensurprisingly found that retro-isomerization of lycopene in isomerizedtomato oleoresin can be minimized by reducing its content in 13-Zisomer.

In an embodiment, the lycopene-containing material can be an extract, aconcentrate or an oleoresin, which is obtained, extracted, enriched orpurified from a plant or vegetable material, a microorganism, a yeast ora product of animal origin. It is further subjected to a treatment toincrease its Z isomer content of lycopene, as described below.

If the source of lycopene is from plant origin, it may be vegetables,leaves, flowers, fruits and other parts of the plant. In a preferredembodiment, the source of lycopene is tomatoes (i.e., whole tomato,tomato extract, tomato flesh, tomato puree, tomato skin, with or withoutthe seeds). Suitable plant or vegetable concentrates are obtainable e.g.by drying or freeze-drying the fresh-cut plants or vegetables or therespective roots, fruits or seeds thereof and then optionally grindingor granulating the dried material. Suitable methods of obtainingextracts of the above-mentioned plants or vegetables are known in theart. The plant or vegetable extracts can be obtained, for example, byextracting the fresh-cut or processed plants or vegetables or therespective roots, fruits or seeds thereof with water or with one or morefood grade solvents or with a mixture of water and one or more foodgrade solvents. Preferably, the extracts and concentrates according tothe present invention may be lipidic or aqueous. Because carotenoids areliposoluble, extraction with water will remove unwanted constituentsthat are water-soluble such as, for example, sugars, amino acids,soluble proteins and/or organic acids.

If the lycopene-containing material is obtained from microorganism, anymicroorganism that produces lycopene may be used, in particularprobiotic microorganism such as, for example, lactic acid bacterium.Also, the product of animal origin may be from, for example, salmon,shrimps, krill or a liver extract or a milk fraction. In the presentspecification, the term “milk fraction” should be understood to mean anypart of the milk.

In an alternative embodiment, the lycopene-containing material can be anoleoresin. Suitable methods for obtaining oleoresins from theabove-mentioned plants or vegetables are well known in the art. Forexample, oleoresins can be obtained by lipidic extraction using asolvent compatible with the food business, cosmetics or pharmaceuticals.Oleoresins prepared by conventional methods have a content in lycopeneof about 0.05% to 50% by weight. Their content of all-E isomer oflycopene is usually higher than that of Z-isomers, e.g. the ratio of Z/Eisomers of lycopene in a selected tomato oleoresin is about 7:93.

Oleoresins are preferred starting material for obtaining the primarycomposition according to the present invention because they containother carotenoids or antioxidants such as Vitamin E, which alsostabilize the composition. The bioactivity and stability of the lycopenecompound in the oleoresin can be improved, in particular, during theisomerization process and the yield of the Z lycopene in the primarycomposition can also be increased.

The lycopene-containing material preferably includes carotenes andxanthophylls such as, for example, zeaxanthine, astaxanthine,beta-cryptoxanthin, capsanthine, canthaxanthine, lutein and derivativesthereof such as esters, for example. The lycopene compounds have beensubjected to a treatment to increase the Z isomer fraction in theprimary composition.

In order to obtain such an isomer profile, the lycopene-containingmaterial which is in the form of an extract, a concentrate or anoleoresin, is subjected to an isomerization by using neutral, acidic orbasic solid catalysts (e.g. clays, zeolites, molecular sieves, ionexchangers) to produce mixtures with high Z/E ratio. The use of solidcatalysts to enrich the lycopene in Z-isomers is not polluting andharmful to the food since the catalysts can be conveniently removed bysimple filtration or centrifugation. Also, combinations of solidcatalysts with other common means (e.g. heat, light and radicalinitiators) can further enhance the geometrical isomerization.

In another embodiment, the extracts or derivatives according to theinvention can be prepared starting from tomatoes, parts of tomatoes(such as the skin), derivatives (such as sauces and concentrates) orextracts. Isomerization is carried out by prolonged heating in asolvent. This finding is surprising in view of contrary teachings whichmay be derived from the prior art discussed in the section “Backgroundof the Invention”.

In particular, when tomatoes or derivatives thereof are used as startingmaterials, they can be treated with a solvent able to extract lycopene.The resulting extract is then heated, the solvent is removed, thusrecovering the isomerized extract.

On the other hand, when an extract or derivative is used as startingmaterial, this is taken up in a solvent, the mixture is heated for asuitable time, then the solvent is removed, thus recovering theisomerized extract. Solvents which can be used for the isomerizationstep are hydrocarbons, chlorinated hydrocarbons, esters, ketones,alcohols; particularly C3-C10 aliphatic hydrocarbons, C1-C3 chlorinatedsolvents, C3-C6 esters, C3-C8 ketones and C1-C8 alcohols; moreparticularly hexane, carbon tetrachloride, ethyl acetate, acetone andbutanol. Isomerization in solvents is carried out at temperaturesranging from 50 to 150° C., preferably at temperatures ranging from 60to 130° C. Isomerization time ranges from 4 to 240 h, preferably from 10to 180 h.

The Z/E isomer ratio in the primary composition may then be increased upto at least 20:80, preferably between 20:80 and 95:5, more preferablyfrom 30:70 to 90:10. In a preferred embodiment, the (5Z+9Z)/E ratio isabove 1, and the 13Z is partly removed.

In an embodiment, the present invention provides a primary composition,in the form of a powder, liquid or gel, comprising a lycopene compoundwhich has a better bioavailability and/or bioefficacy than the compoundalone. Also, the primary composition may be in the form of a highlywater-dispersible composition, if the powder form is chosen. In thisinstance, the powder is dispersible in water at ambient temperature. Theprimary composition also provides carotenoids in a particularly highlysoluble form in lipids and organic solvents, less prone tocrystallization, and having a lower tendency to aggregate.

In another embodiment of the present invention, the primary compositionmay be used either alone or in association with other active compoundssuch as vitamin C, vitamin E (tocopherols and tocotrienols), carotenoids(carotenes, lutein, zeaxanthine, beta-cryptoxanthine, etc.) ubiquinones(e.g. CoQ10), catechins (e.g. epigallocatechin gallate), coffee extractscontaining polyphenols and/or diterpenes (e.g. kawheol and cafestol),extracts of chicory, Ginkgo biloba extracts, grape or grape seedextracts rich in proanthocyanidins, spice extracts (e.g. rosemary), soyextracts containing isoflavones and related phytoestrogens and othersources of flavonoids with antioxidant activity, fatty acids (e.g. n-3fatty acids), phytosterols, probiotic fibers, probiotic microorganisms,taurine, resveratrol, aminoacids, selenium and precursors ofglutathione, or proteins such as, for example, whey proteins.

The primary composition can additionally comprises one or more ofemulsifiers, stabilizers and other additives. Emulsifiers compatible inthe food field are, for example, phospholipids, lecithin,polyoxyethylene sorbitan mono- or tristearate, monolaurate,monopalmitate, mono- or trioleate; a mono- or diglyceride. Any type ofstabilizer that is known in the food business, in cosmetics or inpharmaceuticals can be added. Also, flavours, colorants and any othersuitable additives known in the food business, in cosmetics or inpharmaceuticals can be added. These emulsifiers, stabilizers andadditives can be added according to the final uses of the primarycompositions.

In an alternative embodiment, the present invention provides an oralcomposition comprising the primary composition described above in afoodstuff, in a food supplement, in a pet food product, in a cosmeticpreparation or in a pharmaceutical preparation.

In a preferred embodiment, a food composition for human consumption canbe supplemented by the primary composition. This food composition maybe, for example, a nutritional complete formula, a dairy product, achilled or shelf stable beverage, a mineral water, a liquid drink, asoup, a dietary supplement, a meal replacement, a nutritional bar, aconfectionery, a milk or a fermented milk product, a yoghurt, a milkbased powder, an enteral nutrition product, an infant formulae, aninfant nutritional product, a cereal product or a fermented cereal basedproduct, an ice-cream, a chocolate, coffee, a culinary product such asmayonnaise, tomato puree or salad dressings or a pet food.

For use in food compositions, the primary composition can be added tothe above-mentioned foods or drinks so as to have a daily intake betweenabout 0.001 and 50 mg of lycopene contained in the primary composition.A daily intake of about 5 to 20 mg per day is preferably envisaged.

The nutritional supplement for oral administration may be in capsules,gelatin capsules, soft capsules, tablets, sugar-coated tablets, pills,pastes or pastilles, gums, or drinkable solutions or emulsions, syrupsor gels, with a dose of about 0.001% to 100% of the primary composition,which can then be taken directly with water or by any other known means.This supplement may also include a sweetener, a stabilizer, an additive,a flavour or a colorant. A supplement for cosmetic purposes canadditionally comprise a compound active with respect to the skin. Itshould be appreciated that the supplements can be made by any methodsknown by those skilled in the art.

In another embodiment, a pharmaceutical compositions containing theprimary compositions can be administered for prophylactic and/ortherapeutic treatments, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Inthe present specification, an amount adequate to accomplish this isdefined as “a therapeutically effective dose”. Amounts effective forthis will depend on the severity of the disease and the weight andgeneral state of the patient.

In prophylactic applications, primary compositions according to theinvention can be administered to a patient susceptible to or otherwiseat risk of a particular disease. Such an amount is defined to be “aprophylactic effective dose”. In this use, the precise amounts againdepend on the patient's state of health and weight.

In an alternative embodiment, the primary compositions of the inventioncan be administered with a pharmaceutical acceptable carrier, the natureof the carrier differing with the mode of administration, for exampleparenteral, intravenous, oral and topical (including ophthalmic) routes.The desired formulation can be made using a variety of excipientsincluding, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin cellulose, magnesiumcarbonate. The pharmaceutical compositions may be a tablet, a capsule, apill, a solution, a suspension, a syrup, a dried oral supplement, a wetoral supplement.

Preferably, for humans the pharmaceutical compositions according to thepresent invention can comprise an amount of the primary composition asdescribed above, for a daily administration, so that the lycopene amountranges from about 0.01 mg to 100 mg. When administered daily to pets,the lycopene amount can range from about 0.01 mg to 100 mg.

It will be appreciated that the skilled person will, based on his ownknowledge, select the appropriate components and galenical form totarget the active compound to the tissue of interest, e.g. the skin,colon, stomach, kidney or liver, taking into account the route ofadministration which may be by way of injection, topical application,intranasal administration, administration by implanted or transdermalsustained release systems, and the like.

In another embodiment, the present invention provides a cosmeticcomposition comprising the primary composition described above. It maybe formulated in lotions, shampoos, creams, sun-screens, after-suncreams, anti-aging creams and/or ointments, for example. Preferably, thecontent of primary composition can be between 10⁻¹⁰% and 10% by weightof the cosmetic compositions. More preferably, the cosmetic compositionscomprise between 10⁻⁸% and 5% by weight of lycopene. The cosmeticcompositions that can be used topically can additionally comprise a fator an oil which can be used in cosmetics such as, for example, thosementioned in the CTFA work, Cosmetic Ingredients Handbook, Washington.

The cosmetic compositions of the present invention can also include anyother suitable cosmetically active ingredients. The compositionadditionally comprises a structuring agent and an emulsifier. Otherexcipients, colorants, fragrances or opacifiers can also be added to thecosmetic compositions. It will be appreciated that the present cosmeticproducts will contain a mixture of different ingredients known to theskilled person, ensuring a fast penetration of the objective substanceinto the skin and preventing degradation thereof during storage.

It should also be understood that the concepts of the present inventionmay likewise be applied as an adjuvant therapy assisting in presentlyused medications. Because the primary compounds of the present inventionmay easily be administered together with food material, special clinicalfood may be applied containing a high amount of the primarycompositions. It should be clear that on reading the presentspecification together with the appending claims the skilled person willenvisage a variety of different alternatives to the alternativeembodiments mentioned herein.

The present invention additionally relates to the use of the primarycomposition, or the oral composition or the cosmetic compositiondescribed above for the preparation of a product intended to protect thetissues of the skin against aging, in particular for inhibiting damageto the skin and/or mucous membranes by inhibiting collagenases andenhancing the synthesis of collagen. In fact, the use of the primarycomposition as described above, for example, makes it possible toenhance the bioavailability of the lycopene compound in the body and toslow down the aging of the skin. The primary compositions may also beuseful in the prevention or treatment of sensible, dry or reactiveskins, or for improving skin density or firmness, for ameliorating skinphotoprotection, for preventing or treating cardiovascular diseases ordisorders and cancers. They have also particular benefits on hair andcoat of pet animals, such as an improved hair or coat density, fibrediameter, colour, oilness, glossiness and a help to prevent hair or coatloss.

The positive effects of the primary composition of the present inventionon the skin of humans or pets can be measured by using conventionalmethods such as, for example, minimal erythemal dose (MED), colorimetry,transepidermal water loss, DNA repair, measure of interleukins andproteoglycans production, or collagenase activity, barrier function orcell renewal or ultrasonic echography.

EXAMPLES Example 1 Study of the Stability of Lycopene Isomers

The stability of lycopene isomers was evaluated both in an organicsolvent and in a tomato extract.

Materials

Lycopene-rich tomato oleoresin has been obtained from Indena s.p.a.(Milan, Italy). Its total lycopene content amounted to 9.1%, of whichthe all-E and the 5-Z isomers represented 93.5% and 6.5%, respectively.Two isomerized oleoresins were prepared by heating a suspension oftomato oleoresin in ethyl acetate (1:10 w/w) either for 1 h or for 48 h.After cooling at room temperature, the suspensions were centrifuged andethyl acetate in the recovered supernatants was removed by distillationunder reduced pressure.

Di-t-butyl-hydroxy-toluene (BHT) and N-ethyldiisopropylamine were fromFluka AG. All solvents were HPLC grade and were used withoutpurification.

Isolation of Pure Lycopene Isomers

Pure 5-Z, 9-Z, 13-Z and all-E lycopene were isolated from isomerisedtomato oleoresin (submitted to 1 hour heating), by collecting thefractions containing the corresponding peaks after HPLC separation (seebelow the experimental conditions). Peaks were collected during twoconsecutive HPLC runs and the corresponding fractions were pooled.

Lycopene Analysis

Amount of total lycopene was determined by reverse phase HPLC on a C₁₈precolumn (ODS Hypersil, 5 μm, 20×4 mm; Hewlett Packard, Geneva,Switzerland) and a C₁₈ column (Nova pak, 3.9 μm i.d.×300 mm length,Millipore, Volketswil, Switzerland). The separation was achieved at roomtemperature under isocratic conditions with a mobile phase consisting ofacetonitrile/tetrahydrofuran/methanol/ammonium acetate 1%(533.5:193.6:53.7:28, wt/wt/wt/wt). The mobile phase flow rate was 1.5mL/min.

Lycopene isomer profiles were determined by normal phase HPLC accordingto the method described by Schierle et al. (Schierle, J., Bretzel, W.,Buhler, I., Faccin, N., Hess, D., Steiner, K., Schuep, W. (1997). Food.Chem. 59: 459). Samples of isomerized oleoresins were dissolved inn-hexane containing 50 ppm BHT and spun at maximum speed in an EppendorfLab centrifuge. The resulting supernatants were immediately analyzed byHPLC. The HPLC system used was a 1100 series Hewlett-Packard modelequipped with an ultraviolet-visible photodiode array detector. Datawere simultaneously acquired at 470 nm, 464 nm, 346 nm and 294 nm.Samples (10 μL) were separated using a combination of three Nucleosil300-5 columns (4 mm internal diameter×250 mm length, Macherey-Nagel).The separation was achieved at room temperature under isocraticcondition with a mobile phase consisting of n-hexane with 0.15%N-ethyldiisopropylamine. Flow rate was 0.8 mL/min. Lycopene Z-isomerswere identified according to literature data.

Amounts of lycopene isomers were calculated based on surface areas ofthe HPLC peaks using the same extinction coefficient as the all-Elycopene. Therefore, the lycopene concentration in products containingZ-isomers is slightly underestimated since it is recognized that theextinction coefficients of Z-isomers are lower than that of the all-Eisomer.

Conditions for Stability Tests

Stability of lycopene isomers was investigated both in n-hexane and in atomato oleoresin isomerized by 4 hour heating in ethyl acetate. For thispurpose, pure lycopene isomers were stored for 33 days in n-hexane atroom temperature and in the absence of light, and the isomerized tomatooleoresin was kept for 55 days at room temperature in the absence oflight. Total lycopene concentration and lycopene isomer profiles weremeasured at various time intervals during the storage.

Results

Stability of Lycopene Isomers in N-Hexane

Results of the stability test of pure lycopene isomers during storage inn-hexane at room temperature in the absence of light are reportedTable 1. All isomers, i.e. included the all-E isomer, underwent ageometrical isomerization during storage. The 13-Z was the less stableisomer: whereas less than 50% of 5-Z, 9-Z and all-E lycopene weretransformed after 33 day storage, more than 80% of 13-Z lycopene wasconverted into other isomers during this period of time. Also, thetransformation pathway was different for the 13-Z lycopene compared tothe other Z-isomers: while the 13-Z isomer was mainly converted into theall-E isomer, the 5-Z and 9-Z isomers were principally transformed intoother Z-isomers during storage in n-hexane.

TABLE 1 Stability of pure lycopene isomers in n-hexane during storage atroom temperature concentration (% of total isomers) time (days) all E13-Z 9-Z 5-Z x-Z all-E 0 97.6 1.4 0.5 0.5 0.1 lycopene 1 86.0 10.1 1.21.2 1.5 2 78.4 15.0 1.1 2.6 3.0 5 69.3 19.6 2.2 3.8 5.1 12 67.8 18.2 2.06.4 5.6 33 58.7 15.7 3.8 13.4 8.4 5-Z 0 1.1 n.d. n.d. 95.5 3.4 lycopene1 2.2 n.d. n.d. 84.3 13.5 2 2.4 n.d. n.d. 76.9 20.6 5 3.9 n.d. n.d. 68.427.7 12 5.6 1.7 0.7 65.3 26.7 33 10.7 2.8 2.3 53.5 30.7 9-Z lycopene 04.4 0.6 93.3 1.8 0 1 5.8 2.3 87.1 2.1 2.8 2 6.0 3.1 83.5 11.6 5.9 5 5.65.2 79.2 1.5 8.6 12 7.0 8.2 66.5 2.3 15.9 33 9.8 10.5 56.0 4.1 19.7 13-Z0 2.4 96.6 0 0 1.0 lycopene 1 42.6 57.0 0.4 0 0 2 59.8 38.7 0 0 1.5 568.9 23.4 1.5 1.9 4.3 12 65.5 20.8 2.6 5.3 5.8 33 57.0 16.9 4.2 11.710.2

Stability of Lycopene Isomers in Tomato Oleoresin

Results of the stability test of lycopene isomers in a tomato oleoresinheated for 48 hours in ethyl acetate are reported in Table 2.

TABLE 2 Stability of lycopene isomers in isomerized tomato oleoresinduring storage at room temperature (n = 2). Storage Total time lycopene(days) (mg/g) 13-Z % 9-Z % all-E % 5-Z % 0 055.6 ± 3.0  17.4 ± 0.4  32.7± 0.7 18.7 ± 0.6 12.0 ± 0.4 3 56.6 ± 0.8 12.4 ± 0.1  31.4 ± 0.3 25.6 ±0.5 13.0 ± 0.1 5 58.7 ± 0.4 9.5 ± 0.0 30.6 ± 0.5 30.6 ± 0.6 14.0 ± 0.4 759.2 ± 0.1 7.3 ± 0.2 30.9 ± 0.7 32.9 ± 0.3 14.4 ± 0.2 11 59.3 ± 0.8 5.2± 0.1 29.4 ± 0.0 36.5 ± 0.1 15.0 ± 0.1 17 60.1 ± 0.9 3.3 ± 0.4 29.0 ±0.4 38.9 ± 0.4 15.4 ± 0.4 20 58.8 ± 0.9 2.9 ± 0.3 29.2 ± 0.4 39.9 ± 1.315.2 ± 0.6 34 51.3 ± 7.7 2.2 ± 0.1 30.0 ± 0.1 40.1 ± 0.7 14.1 ± 1.0 4753.9 ± 1.2 1.9 ± 0.6 30.0 ± 0.3 41.4 ± 0.7 14.6 ± 1.3

Total lycopene content was stable during storage at room temperature.However, the lycopene isomer profile markedly changed with a decrease of13-Z lycopene content and an increase of the all-E lycopene. The contentof 9-Z and 5-Z lycopene remained stable during the storage period.

Conclusion

Both stability tests have shown that the 13-Z lycopene was much lessstable than either the 5-Z, or the 9-Z, or the all-E isomers.Consequently, an isomerized tomato oleoresin with a low level of 13-Zlycopene should exhibit a good stability of its lycopene isomer profile.

Example 2 Isomerized Tomato Oleoresin with Increased Bioavailability

Objective:

The objective of the present work was to investigate the bioavailabilityof various Z-lycopene isomers in humans. To elucidate thebioavailability of specific Z-lycopene isomer in human, tomatooleoresins have been enriched in different Z-lycopene isomers reachingabout 60% of the content of total lycopene i.e. one rich in 5-Zlycopene, another one rich in 13-Z lycopene and the last one rich in amixture of 9-Z lycopene and 13-Z lycopene.

Material and Method

Subject

Thirty healthy men were enrolled in the study. The inclusion criteriawere that the subjects should be nonvegetarians and nonsmokers and thatthey have no metabolic disorders such as diabete; hypertension; renal,hepatic, or pancreatic disease; or ulcers. Subjects were normolipidemic,i.e. they had a ratio of plasma cholesterol to HDL cholesterol <5.0 andplasma triacylglycerol (TAG) concentrations <1.5 mmol/L. Because of thelarge amount of blood that was drawn during the study, subjects wererequired to have a blood hemoglobin concentration >13 g/dL. Subjectswere excluded from the study if they used cholesterol-alteringmedication or hypolidemic treatment or vitamin and mineral supplementsfrom 3 months before the start of the study until the completion of thestudy or had had major gastrointestinal surgery; exercised intensively,such as running marathons; and consumed daily >2 glasses of wine (3dL), >2 beers (3 dL), or >1 glass (shot glass) of hard liquor.Twenty-seven of the 30 volunteers completed the 4 post-prandial tests.Three volunteers abandoned the trial before the end for the followingreasons: unavailability, medical treatment related to an eye injury,nausea related to the consumption of fatty meals. Subjects were 24±1 yold with a body weight of 70±1 kg and body mass index (BMI) of 22.5±0.3kg/cm².

The protocol was approved by the ethical committee of Marseille(Marseille, France). Subjects received information on the background anddesign of the study and gave written informed consent beforeparticipation. They were free to withdraw from the study at any time.

Study Design

This was a double-blind, randomized, 4-periods, 4-treatments cross-overclinical trial with a washout period of 3 weeks minimum. After anovernight fast, subjects arrived at the Clinical Pharmacology andTherapeutic Trial Center of University of Marseille and consumed astandard meal consisting of 25 mg lycopene incorporated in 40 g peanutoil that was mixed with 70 g wheat semolina (cooked with 200 mL tapwater). In addition, they consumed 40 g bread, 60 g cooked egg whites, a125 g yoghurt containing 5 g of white sugar and drank 330 mL of water(Aquarel, Nestle). This standard meal provided 842 kcal (3520 kJ) withthe following nutrient composition: protein (11.7%), carbohydrates(39.3%) and lipids (49.0%). This meal was consumed within 15 min. Noother food was allowed over the subsequent 6 h, but subjects wereallowed to drink up to a bottled water (330 ml) during the last 3 hpost-absorption (Aquarel, Nestle).

Lycopene Supplements

Four different tomato products were tested providing each one 25 mg oftotal lycopene. They consisted of:

-   -   Tomato paste (Thorny, Switzerland) containing lycopene mostly in        all-E configuration    -   Tomato oleoresin enriched in 5-Z lycopene    -   Tomato oleoresin enriched with 13-Z lycopene

Tomato oleoresin enriched with a mixture of 9-Z and 13-Z lycopene

Table 3 presents the lycopene content as well as the lycopene isomerprofile of these four tomato products.

TABLE 3 Total lycopene as well as all-E and sum of Z-lycopene isomers inthe four tomato products All-E 5-Z 9-Z 13-Z X*-Z (% of total (% of total(% of total (% of total (% of total lycopene) lycopene) lycopene)lycopene) lycopene) Tomato 94.9 4.1 nd  0.1 nd paste 5-Z 33.4 65.3 1.3nd nd 13-Z 29.3 7.6 9.6 41.5 12.0 9- & 13-Z 27.7 7.7 30.8 23.5 10.2*unidentified lycopene isomers is a pool of unknown lycopene isomerscalculated from the corresponding peak areas in the HPLC chromatogram.

Collection of Blood Samples

Fasting blood was drawn from an anticubital vein by venipuncture into anevacuated tube containing potassium EDTA/K₃ that was immediately placedin an ice-water bath and covered with an aluminium foil to avoid lightexposure. Fasting blood samples were collected before i.e. 20 minutesand 5 minutes before consumption of the standard meal as well as 2 h, 3h, 4 h, 5 h, 6 h post-absorption. The tube containing the blood wasprotected from light, stored at 4° C. and then centrifuged within 2 h(10 min, 4° C., 2.800 rpm) to separate the plasma. A cocktail ofinhibitors (10 μL/mL) was added (Cardin et al., Degradation ofapolipoprotein B-100 of human plasma low density lipoproteins by tissueand plasma kallikreins, Biol Chem 1984; 259:8522-8.).

Isolation of Plasma Triglyceride-Rich Lipoproteins (TRL)

After consumption of a fatty meal, dietary lipophilic molecules areincorporated into chylomicrons, which are secreted into blood.Lipoproteins are separated by ultracentrifugation methodology based ontheir density. Due to the quite similar density of chylomicrons (0.95g/ml) and VLDL (1.006 g/ml), it is not possible to separate one from theother and they are collected altogether in a fraction calledtriglyceride-rich lipoproteins (TRL). However, in the post-prandialstate, this plasma TRL fraction contains mainly chylomicrons secretedfrom the intestine, which is a good assessment of the intestinalbioavailability.

Triglyceride-rich lipoproteins (TRL) containing mainly chylomicrons withlittle amount of VLDL were immediately isolated by ultracentrifugationas follows: 6 mL of plasma were overlaid with a 0.9% NaCl solution andultracentrifuged for 28 min at 32.000 rpm, at 10° C. in a SW41TI rotor(Beckman), in a L7 ultracentrifuge (Beckman). Immediately aftercentrifugation, the TRL were aliquoted and stored at −80° C. beforeanalytical determinations. Lycopene analyses were performed within 10days, and triacylglycerol analyses within 30 days.

Analytical Determination

Triglycerides were assayed by an enzymatic and colorimetric method usinga commercial kit (Kit Bio-Merieux).

Total lycopene and lycopene isomer profiles were determined by reversephase and normal phase HPLC method, respectively (M. Richelle, K.Bortlik, S. Liardet, C. Hager, P. Lambelet, L. A. Applegate, E. A.Offord, J. Nutr. (2002) 132, 404-408.). Total lycopene content wascalculated as the sum of the 5-Z, 9-Z, 13-Z, x-Z and all-E-lycopeneisomers. Lycopene isomer was quantified using the extinction coefficientof all-E lycopene since the exact value for all individual Z-lycopene isstill unknown. Profile of lycopene isomers is determined by the ratio ofindividual lycopene isomer to total lycopene expressed in percentage.

Statistical Analysis

Lycopene bioavailability was assessed by measuring the area under thelycopene concentration in TRL-time curve (AUC). This area was calculatedover the 0-6 hour period using the trapezoidal method (AUC(0-6 h)). Dataare presented as mean±SEM. The baseline concentration was the average ofthe concentrations measured in the two plasma samples collected beforeconsumption of the standard meal containing 25 mg lycopene from thetomato matrix. For each subject and each lycopene treatment, calculationof the AUC(0-6 h) was performed by subtracting the baselineconcentration from the concentration value measured at each time pointpost-absorption. If this value was negative, it was considered as zero.

For each treatment, if the distribution of the AUC_((0-6h)) was normal(Skewness and Kurtosis tests) with or without logarithmictransformation, comparison was performed by using a linear mixed modelwith treatment as fixed effect and subject as random effect.

All statistical analyses were done with SAS software (version 8.2; SASInstitute, Cary, N.C.). The rejection level in statistical tests wasequal to 5%.

Results

Lycopene Bioavailability

Because the four tomato treatments induced a variation of the extent intriglyceride secretion, lycopene bioavailability has been normalizedusing triglyceride absorption (AUC_((0-6h))).

Normalized lycopene bioavailability was markedly different between thefour tomato treatments (FIGURE).

Surprisingly, lycopene was better bioavailable, by about two times, fromtomato oleoresin rich in 5-Z lycopene than from the other threetreatments, i.e tomato paste, tomato oleoresin rich in 13-Z lycopene aswell as tomato oleoresin rich in a mixture of 13-Z and 9-Z lycopene(p<0.0001) (FIGURE).

While lycopene was similarly bioavailable from tomato paste as from themixture of 13-Z and 9-Z tomato oleoresin.

Lycopene present in 13-Z tomato oleoresin exhibited a slight butsignificant lower bioavailability (p<0.03) compared to tomato paste.

Conclusion

These results indicate that the configuration of the lycopene moleculeaffects markedly the trafficking of lycopene within the gastrointestinaltract and in consequence the amount of lycopene that is absorbed.Lycopene bioavailability from tomato extract rich in 5-Z lycopene isabout double than that from tomato paste. In contrast, lycopene presentin tomato extract rich in a mixture of 9-Z and 13-Z lycopene issimilarly bioavailable to that present in tomato paste while tomatooleoresin rich in 13-Z lycopene presents a slightly less bioavailablelycopene. Several authors have already pointed out that the presence ofZ-lycopene in a tomato product is associated with an increase oflycopene bioavailability. This is the first study demonstrating that theenhancement of lycopene bioavailability is specifically related tolycopene configuration, i.e. 5-Z lycopene >9-Z lycopene >13-Z lycopene.

Example 3 Extraction and Isomerization in Ethyl Acetate

52 kg of fresh tomatoes containing 100 ppm of lycopene are chopped andhomogenized. Part of the water is distilled off under reduced pressureto obtain 18 kg of tomato concentrate. This is extracted with 36 l ofwater saturated ethyl acetate; during extraction, the mixture is kept atroom temperature shielded from light and under stirring for 2 hours. Theextract is then separated from the tomato concentrate. The abovedescribed procedure is repeated twice on such tomato concentrate,totally using 108 l of solvent. The combined extracts are washed in aseparatory funnel with 27 l of water. The aqueous phase is thendiscarded while the organic phase is concentrated under reduced pressureto obtain a suspension with 10% w/v dry residue; the dry residue has atotal lycopene content of 9.1% w/w and a Z isomer content of 0.46% w/w.This mixture is refluxed (76° C.) under stirring for 7 days before beingconcentrated to dryness under reduced pressure.

46.8 g of final extract with a total lycopene content of 9% w/w and a Zisomer content of 5.59% w/w are obtained; in particular, the E isomercontent is 3.41% w/w and the 13-Z isomer content is 0.16% w/w. The HPLCprofile of the extract is reported in the FIGURE.

Example 4 Extraction and Isomerization in Hexane

10 kg of fresh tomatoes containing 140 ppm of lycopene are chopped andhomogenized. Part of the water is distilled off under reduced pressureto obtain 2.5 kg of tomato concentrate, which is extracted with 12.5 lof hexane. During extraction, the mixture is kept at room temperatureshielded from light and under stirring for 2 hours. The extract is thenseparated from the tomato concentrate. The above described procedure isrepeated once on such tomato concentrate, totally using 25 l of solvent.The extracts are combined and concentrated under reduced pressure toobtain a solution with 10% w/v dry residue; the dry residue has a totallycopene content of 9.1% w/w and a Z isomer content of 0.46% w/w. Thismixture is refluxed (69° C.) under stirring for 6 days before beingconcentrated to dryness under reduced pressure. 16.5 g of final extractwith total lycopene content of 9.1% w/w and Z isomer content of 5.62%w/w are obtained; in particular, the E isomer content is 3.38% w/w andthe 13-Z isomer content is 0.18% w/w.

Example 5 Isomerization in Butanol

10 kg of fresh tomatoes containing 90 ppm of lycopene are chopped andhomogenized. Part of the water is distilled off under reduced pressureto obtain 3.4 kg of tomato concentrate, which is extracted with 7 l ofwater-saturated ethyl acetate. During extraction, the mixture is kept atroom temperature shielded from light and under stirring for 2 hours. Theextract is then separated from the tomato concentrate. The abovedescribed procedure is repeated twice on such tomato concentrate,totally using 21 l of solvent. The combined extracts are washed in aseparatory funnel with 5.3 l of water. The aqueous phase is thendiscarded while the organic phase is concentrated to dryness underreduced pressure. The dry residue (9.8 g), which has a total lycopenecontent of 7.8% w/w and a Z-isomer content of 0.40% w/w, is suspended in98 ml of n-butanol. The mixture is kept at 130° C. under stirring for 4hours before being concentrated to dryness under reduced pressure. 9.8 gof final extract with a total lycopene content of 6.35% w/w and aZ-isomer content of 4.50% w/w are obtained; in particular, the E-isomercontent is 1.85% w/w and the 13-Z isomer content is 0.47% w/w.

Example 6 Isomerization on Solid Catalysts

Materials

Lycopene-rich tomato oleoresin has been obtained from Indena s.p.a.(Milan, Italy). Its total lycopene content amounted to 9.1%, of whichthe all-E and the 5-Z isomers represented 93.5% and 6.5%, respectively.

Methods

A suspension of tomato oleoresin in acetyl acetate (1:100 w/w) wasfiltered and incubated with 5% of solid catalyst under constant stirringat room temperature for 2 h. The mixture was centrifuged at maximumspeed in an Eppendorf Lab centrifuge and an aliquot of supernatantevaporated under N₂ and re-suspended in n-hexane/BHT.

Lycopene Analysis

Amount of total lycopene and lycopene isomer profiles were determined byreverse phase and normal phase HPLC, respectively, under the analyticalconditions described in example 1.

Results

Lycopene isomer profiles measured in tomato oleoresin isomerised for 2 hat room temperature using solid catalysts are reported in Table 4.

TABLE 4 Lycopene isomer profiles in tomato oleoresins isomerised usingsolid catalysts Isomer concentration (% of total isomers) Catalyst All-E13-Z 9-Z 5-Z x-Z* control 83.3 3.0 0.9 8.6 4.1 Tonsil Optimum 31.3 7.013.4 23.8 24.5 Amberlyst 15 34.5 4.8 11.2 19.4 30.1 *unknown lycopeneisomers

Lycopene was efficiently isomerised during 2 h reaction in ethyl acetateat room temperature in the presence of either Tonsil Optimum orAmberlyst 15. With both catalysts a large fraction of lycopene all-Eisomer was converted into Z-isomers. Among the identified lycopeneisomers, the 5-Z was majoritairement formed, followed by the 9-Z and the13-Z, respectively; thus, concentration of the 13-Z isomer was, thus,below 10% in the isomerised tomato oleoresins.

1. A method of manufacturing a stable composition enriched incis-lycopene (Z isomers) by prolonged heating in solvent of lycopenecontaining materials.
 2. The method of claim 1 in which thelycopene-containing material consists of tomatoes, parts of tomatoes,derivatives thereof or tomato extracts in solvents.
 3. The method ofclaim 1 wherein the solvent is a C3-C10 aliphatic hydrocarbon, a C1-C3chlorinated solvent, a C3-C6 ester, a C3-C8 ketone or a C1-C8 alcohol.4. The method of claim 3 wherein the solvent is selected from hexane,carbon tetrachloride, ethyl acetate, acetone, butanol.
 5. The method ofclaims 1 wherein the heating temperature ranges from 50 to 150° C. 6.The method of claims 1 wherein the heating time ranges from 4 to 240 h.